The importance of the innate immune response as both the first line of defense against infection and a powerful trigger of adaptive immunity, and the definition of signaling pathways involved with this response, have led to intense testing of individual members of these pathways as molecular adjuvants. However, in the case of viral infection, the complete innate immune response is triggered by the presence of viral particles and their constituent components. Our approach is based on the idea that the viral particle itself, if it could be made safe and applicable to a wide range of vaccine modalities, would be a broad and effective inducer of innate immunity, in other words, an excellent adjuvant. Our studies to date indicate that propagation-defective Venezuelan equine encephalitis virus (VEE) replicon particles safely exhibit potent systemic and mucosal adjuvant activity when administered with subunit or inactivated vaccines. To build on these results with the goal of producing a safe and effective second generation adjuvant for biodefense, we propose the following four aims: Aim 1 - to further define VEE adjuvant activity in mice;Aim 2 - with our collaborators, to test adjuvant activity for specific biodefense vaccines, including avian influenza, poxviruses, Rift Valley Fever virus, Guanarito and Junin hemorrhagic fever viruses, and F. tularensis;Aim 3 - in collaboration with the Center for Comparative Medicine at U.C.-Davis, to determine the level of adjuvant activity in primates;and Aim 4 - to develop a scalable manufacturing process for adjuvant production.